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. 2010 Mar 22;188(6):863–876. doi: 10.1083/jcb.200907135

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© 2010 Cain et al.

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Figure 9. — Pyk2 and Rac1 depletion alter endothelial junctions and leukocyte TEM. HUVECs were transfected with either control siRNA or siRNA to Pyk2 and/or Rac1 and then stimulated with TNF (18 h) or left unstimulated. (A) HUVECs were lysed and analyzed by SDS-PAGE and Western blotting with Pyk2, Rac1, and GAPDH antibodies to assess siRNA knockdown efficiency and ICAM-1 to assess TNF stimulation. The asterisk indicates a nonspecific band; Rac1 is the top band. (B) Barrier function of siRNA-treated HUVECs either stimulated with TNF (16–18 h) or left unstimulated was assessed by permeability to FITC-dextran. Unstimulated monolayers were assigned as 100%. (C) Immunofluorescence micrographs of siRNA-transfected HUVECs. Samples were stained with antibodies to VE-cadherin and Alexa Fluor 633–conjugated phalloidin to visualize F-actin. Arrowheads indicate examples of overlapping junctions. (D) THP-1 cells were added to siRNA-treated, TNF-stimulated HUVECs in Transwell chambers, and the resultant TEM efficiency toward MCP-1 was determined after 1 h. (E and F) Junctional index (junctional area/cell number; E) and cell circularity (F) were determined from immunofluorescence images. Data represent the mean and SEM of at least four independent experiments, each performed in triplicate. Statistical significance was assessed by the Mann-Whitney U test; *, P < 0.05; **, P < 0.02. Bars, 20 µm.